title: Quantitative detection of Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa in human oral epithelial cells from subjects with periodontitis and periodontal health.
abstract: Epithelial cells in oral cavities can be considered reservoirs for a variety of bacterial species. A polymicrobial intracellular flora associated with periodontal disease has been demonstrated in buccal cells. Important aetiological agents of systemic and nosocomial infections have been detected in the microbiota of subgingival biofilm, especially in individuals with periodontal disease. However, non-oral pathogens internalized in oral epithelial cells and their relationship with periodontal status are poorly understood. The purpose of this study was to detect opportunistic species within buccal and gingival crevice epithelial cells collected from subjects with periodontitis or individuals with good periodontal health, and to associate their prevalence with periodontal clinical status. Quantitative detection of total bacteria and Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis in oral epithelial cells was determined by quantitative real-time PCR using universal and species-specific primer sets. Intracellular bacteria were visualized by confocal microscopy and fluorescence in situ hybridization. Overall, 33% of cell samples from patients with periodontitis contained at least one opportunistic species, compared with 15% of samples from healthy individuals. E. faecalis was the most prevalent species found in oral epithelial cells (detected in 20.6% of patients with periodontitis, P = 0.03 versus healthy individuals) and was detected only in cells from patients with periodontitis. Quantitative real-time PCR showed that high levels of P. aeruginosa and S. aureus were present in both the periodontitis and healthy groups. However, the proportion of these species was significantly higher in epithelial cells of subjects with periodontitis compared with healthy individuals (P = 0.016 for P. aeruginosa and P = 0.047 for S. aureus). Although E. faecalis and P. aeruginosa were detected in 57% and 50% of patients, respectively, with probing depth and clinical attachment level ≥6 mm, no correlation was found with age, sex, bleeding on probing or the presence of supragingival biofilm. The prevalence of these pathogens in epithelial cells is correlated with the state of periodontal disease.
authors.affiliation: Institute of Biomedical Sciences, Department of Microbiology, University of São Paulo, São Paulo, Brazil.
authors.forename: Andrea V
authors.initials: AV
authors.lastname: Colombo
authors.affiliation: Institute of Biomedical Sciences, Department of Microbiology, University of São Paulo, São Paulo, Brazil.
authors.forename: Graziela M
authors.initials: GM
authors.lastname: Barbosa
authors.affiliation: Institute of Biomedical Sciences, Department of Microbiology, University of São Paulo, São Paulo, Brazil.
authors.forename: Daniela
authors.initials: D
authors.lastname: Higashi
authors.affiliation: School of Dentistry, Department of Periodontology, University of São Paulo, São Paulo, Brazil.
authors.forename: Giorgio
authors.initials: G
authors.lastname: di Micheli
authors.affiliation: Institute of Biomedical Sciences, Department of Microbiology, University of São Paulo, São Paulo, Brazil.
authors.forename: Paulo H
authors.initials: PH
authors.lastname: Rodrigues
authors.affiliation: Institute of Biomedical Sciences, Department of Microbiology, University of São Paulo, São Paulo, Brazil.
authors.forename: Maria Regina L
authors.initials: MRL
authors.lastname: Simionato
mesh_terms: D000328:Adult; D000368:Aged; D001424:Bacterial Infections / Q000453:epidemiology / Q000382:microbiology; D058491:Bacterial Load; D002353:Carrier State / Q000453:epidemiology / Q000382:microbiology; D013293:Enterococcus faecalis / Q000302:isolation & purification*; D004847:Epithelial Cells / Q000382:microbiology*; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D018613:Microscopy, Confocal; D008875:Middle Aged; D009061:Mouth Mucosa / Q000382:microbiology*; D010518:Periodontitis / Q000382:microbiology*; D015995:Prevalence; D011550:Pseudomonas aeruginosa / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D013211:Staphylococcus aureus / Q000302:isolation & purification*